Method of enzyme immobilization on a particulate silica carrier for synthesis inorganic media

ABSTRACT

An immobilized enzyme is prepared for synthesis in a mainly organic medium devoid of free water. In one embodiment, an enzyme and liquid binder are introduced by atomization onto a particulate silica carrier having a particle size below 100 μm in a granulator and simultaneously granulating to form the immobilized enzyme. In another embodiment, a liquid enzyme composition is contacted with the particulate silica carrier to obtain a particulate immobilized enzyme having a particle size below 100 μm, the immobilized enzyme is introduced into granulator, a liquid binder is introduced into the granulator and granulation is carried out. The enzyme is preferably a lipase such as a thermostable lipase, and the immobilized lipase is used for interesterification of fats or synthesis of fatty acid esters.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a 35 U.S.C. 371 national application ofPCT/DK95/00076 filed 21 Feb., 1995, which is incorporated herein byreference.

BACKGROUND OF THE INVENTION

The invention comprises a method for production of an immobilized enzymepreparation, comprising an enzyme applicable for organic synthesis in amainly organic medium devoid of free water, and a use of the immobilizedenzyme preparation. The most common enzyme which is applicable fororganic synthesis in a mainly organic medium devoid of free water islipase. Examples of other enzymes of this kind are proteases, amidases,esterases, oxidoreductases, and nitrilases. In the following theinvention usually will be described with reference to lipase as thepredominant example of an enzyme which is applicable for organicsynthesis in a mainly organic medium devoid of free water. The term"organic synthesis" is to be understood as generally accepted in organicchemistry. Thus, typical examples of organic syntheses included in thescope of the invention are the following: reesterifications,transesterifications, interesterifications, acylations, epoxidations,aminolyses, ammoniolyses, oxidations, and reductions. The term "mainlyorganic medium devoid of free water" is to be understood as a one phasemedium, the organic part of which amounts to at least 50% w/w.

Immobilized lipase preparations are used as catalysts forinteresterification and other fat related processes, e.g. cocoa buttersubstitute production. In case of a batch reaction the catalyst has tobe separated from the reaction mixture for reuse when the reaction isfinished. Thus, a good filtrability of the catalyst is needed forsatisfactory performance.

WO 90/05778 describes a method for production of an immobilized lipasepreparation useable for e.g. margarine production. This preparationcomprises a macroporous silica carrier.

EP 140 542 describes an immobilized lipase preparation forinteresterification of fats. This preparation comprises an anionexchange resin carrier.

Both these prior art immobilized lipase preparations suffer from thedisadvantage that they are expensive. Especially in relation to theproduction of margarine, which is produced in millions of tons per yearon a global basis, it is important to minimize production costs.

Thus, the purpose of the invention is the provision of a method forproduction of a cheap immobilized enzyme preparation which shouldexhibit technical properties equal to or almost equal to the prior artimmobilized enzyme preparations, especially in regard to filtrabilityafter a finished batchwise margarine production and in regard to a lowpressure drop in columns for continuous performance, in case the enzymeis a lipase, and of a use of such immobilized enzyme preparation.

SUMMARY OF THE INVENTION

The method according to the invention for production of an immobilizedenzyme preparation comprising an enzyme applicable for organic synthesisin a mainly organic medium devoid of free water, is characterized by thefact that a liquid enzyme composition and a particulate silica carrierwith a particle size below around 100 μm is used as materials to beintroduced into a granulator or extruder, whereafter a granulation or anextrusion is carried out. The liquid enzyme composition can benon-aqueous, e.g. on alcoholic basis, or aqueous. The particulate silicacarrier can exhibit a broad particle size distribution, e.g. betweenaround 5 μm and 100 μm. In this specification with claims "silica" is tobe understood as either silica or a silicate, e.g. magnesium silicate.It is to be understood that the invention both comprises the situation,where a particulate immobilized lipase composition with a particle sizedistribution similar to the particle size distribution of theparticulate silica carrier is first produced, whereafter the granulationor extrusion is carried out (vide Examples 6 and 7), and the situation,where the production is carried out in one step only (vide Examples 1 to5). Also, it is to be understood that the enzyme may act as a binderduring the granulation or extrusion, and/or that a specific bindingagent can be added, e.g. gelatin or polyvinylpyrrolidone. During themethod according to the invention preferably an atomization has to becarried out, usually an atomization of the liquid enzyme compositionand/or an atomization of the binding agent in liquid form. Also, it isto be understood that the apparatus used in the method according to theinvention is of no special importance to the invention, inasmuch as anygranulator, e.g. a fluid bed spray granulator, or any extruder can beused.

A powdered immobilized lipase preparation on silica basis is described,e.g. in WO 88/02775, page 11, lines 21-24. This immobilized lipasepreparation is completely unfit for both batchwise and continuous fatrelated processes, due to poor filtrability after a batch process andgeneration of a high pressure loss during a continuous column process.

Immobilized lipase preparations are described in EP 579928 and in Appl.Microbiol. Biotechnol. (1988) 28:527-530, but none of these prior artlipase preparations comprise a silica carrier.

In Chem. Abstract Vol. 118 (1993): 55095v an immobilized lipasepreparation on a silica carrier is described. However, the methodaccording to the invention comprising particle size of the carrier andgranulation or extrusion is not described.

Surprisingly it has been found that the immobilized enzyme preparationprepared in accordance with the method according to the invention in thefirst place is dramatically cheaper than the comparable prior artimmobilized enzyme preparations, and in the second place that itexhibits technical properties equal to or almost equal to the prior artimmobilized enzyme preparations, e.g. in regard to filtrability after abatchwise fat related process and generation of a low pressure lossduring a continuous fat related process, if the enzyme is a lipase.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1, which is directly related to Example 9, shows percent conversiondepending on time in relation to an ester synthesis performed as acontinuous column operation.

DESCRIPTION OF THE PREFERRED EMBODIMENTS;

A preferred embodiment of the method according to the invention ischaracterized by the fact that the enzyme is a lipase.

A preferred embodiment of the method according to the invention ischaracterized by the fact that the lipase in the liquid lipasecomposition is a thermostable lipase.

A preferred embodiment of the method according to the invention ischaracterized by the fact that the lipase in the liquid lipasecomposition is produced by cultivation of a microorganism containing agene encoding for and expressing a lipase derived from a strain ofHumicola species, Candida antarctica or Rhizomucor miehei.

A preferred embodiment of the method according to the invention ischaracterized by the fact that the proportion between the amount of theliquid lipase composition and the weight of particulate silica carrieris at least 100,000 LU/g of carrier (dry weight). LU is the lipaseactivity unit defined in AF 95.1/2-GB, which can be obtained on requestfrom Novo Nordisk A/S. The LU assay uses tributyrin as a substrate fordetermination of the lipase activity.

A preferred embodiment of the method according to the invention ischaracterized by the fact that the silica has a purity of at least 50%,preferably at least 75%.

A preferred embodiment of the method according to the invention ischaracterized by the fact that a granulator is used, preferably a highspeed mixer or a mixer granulator.

A preferred embodiment of the method according to the invention ischaracterized by the fact that a liquid composition of a binder,preferably gelatin or polyvinylpyrrolidone, is introduced by atomizationinto the granulator or extruder during the granulation or extrusion.

A preferred embodiment of the method according to the invention ischaracterized by the fact that the granulation or extrusion is carriedout for production of the immobilized lipase preparation with a particlesize distribution corresponding to an amount of at least 90% between 50μm and 2,000 μm.

The use of the immobilized enzyme preparation prepared by means of themethod according to the invention is for the process catalyzed by theenzyme.

The use of the immobilized lipase preparation prepared by means of themethod according to the invention is for fat related processes. It is tobe understood that such fat related processes can be performed batchwiseor continuously. When performed batchwise it has been found that theimmobilized lipase preparation produced by means of the method accordingto the invention exhibits a satisfactory filtrability when the enzymaticprocess has come to an end, and when performed continuously it has beenfound that the immobilized lipase preparation produced by means of themethod according to the invention exhibits a good physical strengthresulting in a satisfactory performance of the column.

A preferred embodiment of the use according to the invention is forinteresterification of fats and is characterized by the fact that liquidfats or fatty mixtures, including free fatty acids or fatty acid esters,are contacted with the immobilized lipase preparation.

A preferred embodiment of the use according to the invention is forsynthesis of glycerides or other fatty acid esters and is characterizedby the fact that mixture of glycerol or substituted glycerols or othertypes of alcohols and free fatty acids is contacted with the immobilizedlipase preparation.

A preferred embodiment of the use according to the invention is forsynthesis of glycolipids. The synthesis of glycolipids with immobilizedlipase reparations in general is described in Bjorkling, F. et al.(1989), J. Chem. Soc., Chem. Commun., p. 934-935.

The invention will be illustrated by the following examples.

All manufacturing examples (1-8) illustrate the batchwise embodiment ofthe method according to the invention. For production in industrialscale ordinarily the continuous embodiment will be preferred. Example 9is a use example.

The use according to the invention is illustrated indirectly in Examples1-8, in consideration of the fact that every BAUN determinationillustrates the use (interesterification) according to the invention.The use according to the invention is illustrated directly in Example 9.

EXAMPLES

EXAMPLE 1

65 g of a powder of synthetic magnesium silicate, Celkate T-21(Manville) was introduced into a high speed mixer with an impeller whichcan be operated with a speed of 900 rpm. 75 g of Humicola lanuginosalipase liquid concentrate (prepared according to Danish Patent No.157560, with Humicola lanuginosa DSM 3819, dry substance content 30%,with an activity of 700,000 LU/ml) was continuously atomized onto thesilica powder over a period of approx. five minutes with runningimpeller. The formed granulate was dried overnight at room temperatureand sieved (300-700 μm). The moisture content was adjusted to 10% andthe sample analyzed to 2.6 BAUN/g. The lipase activity assay expressedin BAUN (Batch Acidolysis Units Novo) measures the initial rate ofincorporation of decanoic acid into high oleate sunflower oil (10%water, 70° C.). A detailed description of the method (MP 9410704) isavailable on request from Novo Nordisk A/S. The assay was performedwithout magnetic stirring, but in a shaking water bath.

EXAMPLE 2

65 g of Celkate T-21 was introduced into a high speed mixer as indicatedin Example 1. 25 g of Humicola lanuginosa lipase liquid concentrate asindicated in Example 1 was continuously atomized onto the powder withrunning impeller. Hereafter, 50 g of the Humicola lanuginosa lipaseliquid concentrate with 3% (w/w) Kollidon K25 polyvinylpyrrolidone(BASF) was atomized onto the powder. The formed granulate was driedovernight at room temperature and sieved (300-700 μm). The moisturecontent was adjusted to 10% and the sample analyzed to 0.5 BAUN/g.

EXAMPLE 3

40 g of a powder of a calcinated diatomaceous earth, Clarcel CBL 3 (CecaS.A.) was introduced into a high speed mixer as indicated in Example 1.11 g of Humicola lanuginosa lipase liquid concentrate as indicated inExample 1 was continuously atomized onto the powder with runningimpeller. Hereafter, 47 g of the Humicola lanuginosa lipase with 3%(w/w) Kollidon K25 was atomized onto the powder with running impeller.The formed granulate was dried overnight at room temperature and sieved(300-700 μm). The moisture content was adjusted to 10% and the sampleanalyzed to 2.4 BAUN/g.

EXAMPLE 4

50 g of Clarcel CBL 3 was introduced into a high speed mixer asindicated in Example 1. 72 g of Humicola lanuginosa lipase liquidconcentrate as indicated in Example 1 with 5% (w/w) gelatin (ASFgelatin, Sanofi Bio-lndustries) was continuously atomized onto thepowder liquid concentrate as indicated in Example 1. The formedgranulate was dried overnight at room temperature and sieved (300-700μm). The moisture content was adjusted to 10% and the sample analyzed to5.1 BAUN/g.

EXAMPLE 5

30 g of Clarcel CBL 3 and 20 g of talc powder was introduced into a highspeed mixer as indicated in Example 1. 20 g of Humicola lanuginosalipase liquid concentrate as indicated in Example 1 was continuouslyatomized onto the powder liquid concentrate as indicated in Example 1.Hereafter, 28 g of the Humicola lanuginosa lipase concentrate with 2%(w/w) Methocel A-15 methylcellulose (Dow) was atomized onto the powderwith running impeller. The formed granulate was dried overnight at roomtemperature and sieved (300-700 μm). The moisture content was adjustedto 10% and the sample analyzed to 7.7 BAUN/g.

EXAMPLE 6

250 g of Celkate T-21 was washed with 3 volumes of 0.1M acetate buffer,pH 4.5, for 30 minutes, followed by vacuum filtration. Humicolalanuginosa lipase concentrate as indicated in Example 1 in an amountcorresponding to 500,000 LU/g of Celkate T-21 was added together with 3volumes of 0.1M acetate buffer, pH 4.5, and stirred for two hours atroom temperature. After vacuum filtration the immobilized lipase wasdried for 24 hours at room temperature, the moisture content adjusted to10% and analyzed to 14.3 BAUN/g. The filtrate contained 27565 kLU,corresponding to an adsorption of 78% (or 390 kLU/g).

65 g of the thus dried immobilized lipase on Celkate T-21 powder wasintroduced into a high speed mixer as indicated in Example 1. 55 g of a5% (w/w) gelatin solution was atomized onto the powder with runningimpeller. Hereafter, 0.1 g of Aerosil 200 silicium dioxide (Degussa) wasadded. The formed granulate was dried at room temperature and sieved(300-700 μm). The moisture content was adjusted to 10% and analyzed to5.9 BAUN/g.

EXAMPLE 7

200 g of Clarcel CBL 3 was washed with 3 volumes of 0.1M acetate buffer,pH 4.5, for 30 minutes, followed by vacuum filtration. Humicolalanuginosa lipase concentrate as indicated in Example 1 in an amountcorresponding to 500,000 LU/g of Clarcel CBL 3 was added together with 3volumes of 0.1M acetate buffer, pH 4.5, and stirred for two hours atroom temperature. After vacuum filtration the immobilized lipase waswashed two times with 2-3 volumes of 0.1M acetate buffer, pH 4.5, andtwo times with deionized water. The filtrates contained 82761 kLU intotal correspsonding to an adsorption of 17% (or 86 kLU/g). Theimmobilized lipase was dried for 24 hours at room temperature andanalyzed to 13.4 BAUN/g.

55 g of the thus washed immobilized lipase on Clarcel CBL 3 powder wasintroduced into a high speed mixer as indicated in Example 1.61 g of asolution containing 2% (w/w) gelatin and 1% (w/w) Methocel A-15methylcellulose (Dow) was continuously atomized onto the powder withrunning impeller. Hereafter, 0.1 g of Aerosil 200 silicium dioxide(Degussa) was added. The formed granulate was dried at room temperatureand sieved (300-700 μm). The moisture content was adjusted to 10% andanalyzed to 8.4 BAUN/g.

Another portion, i.e. 59 g of the thus washed immobilized lipase onClarcel CBL 3 powder was introduced into a high speed mixer as indicatedin Example 1.59 g of a 5% (w/w) gelatin solution was continuouslyatomized onto the powder with running impeller. Hereafter, 0.1 g ofAerosil 200 silicium dioxide (Degussa) was added. The formed granulatewas dried at room temperature and sieved (300-700 μm). The moisturecontent was adjusted to 10% and analyzed to 10.1 BAUN/g.

EXAMPLE 8

This is a manufacturing example as Examples 1-7, but with another lipaseproducing microorganism.

Preparation of Sample 1: 12.9 g of Candida antarctica B lipase freezedried powder with an activity of 250,000 LU/g and 1.4 g of Kollidon K25was dissolved in 51 ml of deionized water. 50 g of Celkate T-21 wasintroduced into a high speed mixer as indicated in Example 1 and theabove indicated solution of Candida antarctica B lipase was continuouslyatomized onto the powder with running impeller. The formed granulate wasdried overnight at room temperature and sieved (300-1000 μm).

Preparation of Sample 2: 12.9 g of Candida antarctica B lipase and 0.86g of Methocel A-15 was dissolved in 51 ml of deionized water. 50 g ofCelkate T-21 was introduced into a high speed mixer and the abovesolution was continuously atomized onto the powder with runningimpeller. The formed granulate was dried overnight at room temperatureand sieved (300-1000 μm).

Preparation of Sample 3: 12.8 g of Candida antarctica B lipase and 0.81g of Kollidon K25 was dissolved in 48 ml deionized water. 50 g ofCelkate T-21 was introduced into a high speed mixer and the abovesolution was continuously atomized onto the powder with runningimpeller. The formed granulate was dried overnight at room temperatureand sieved (300-1000 μm).

EXAMPLE 9

This is a use example with the preparation according to Example 8,associated with FIG. 1.

The three samples described in Example 8 were evaluated in columns bycontinuous synthesis of ethyl glucosid esters (EGE) by reacting ethylglucosid (EG) with decanoic acid.

    ______________________________________                                        Reaction conditions:                                                          ______________________________________                                        Column dimensions:                                                                           diameter = 1.5 cm; length = 20 cm                              Sample size:   5.0 g                                                          Substrate:     Ethyl glucosid*)                                                                              4.98 kg                                                       Decanoic acid   4.92 kg                                                       Tertiary butanol 25%                                                                          3.30 kg                                        Temperature:   60° C.                                                  FIow:          30 g/h                                                         Time:          162 hours                                                      ______________________________________                                         *)Synthesized by reacting ethanol and Dglucose in the presence of a catio     exchanger                                                                

Samples were taken after 18, 44, 90, and 162 hours and the content ofEGE and EG was measured on HPLC and % conversion calculated. The resultsare shown in FIG. 1. Moreover, it was noticed that the physicalstability of the samples was good.

We claim:
 1. A method for producing an immobilized enzyme preparationapplicable for organic synthesis in a mainly organic medium devoid offree water, comprising:introducing an enzyme and a binder in liquid formby atomization onto a particulate silica carrier having a particle sizebelow 100 μm in a granulator, and simultaneously carrying out agranulation to form the immobilized enzyme preparation.
 2. The methodaccording to claim 1, wherein the enzyme is a lipase.
 3. The methodaccording to claim 2, wherein the lipase is a thermostable lipase. 4.The method according to claim 2, wherein the lipase is derived from astrain of Humicola species, Candida antarctica or Rhizomucor miehei. 5.The method according to claim 2, wherein the ratio between the lipaseand the particulate silica carrier corresponds to an activity of atleast 100,000 LU/g of carrier (dry weight).
 6. The method according toclaim 1, wherein the silica has a purity of at least 50%.
 7. The methodaccording to claim 6, wherein the silica has a purity of at least 75%.8. The method according to claim 1, wherein the granulator is a highspeed mixer or a mixer granulator.
 9. The method of claim 1, wherein thebinder is gelatin or polyvinyl pyrrolidone.
 10. A method for producingan immobilized enzyme preparation applicable for organic synthesis in amainly organic medium devoid of free water, comprising:contacting aliquid enzyme composition with a particulate silica carrier having aparticle size below 100 μm to obtain a particulate immobilized enzymepreparation having a particle size below 100 μm, and introducing theparticulate immobilized enzyme preparation into a granulator,introducing a liquid comprising a binder by atomization into thegranulator, and carrying out a granulation.
 11. The method according toclaim 10, wherein the enzyme is a lipase.
 12. The method according toclaim 11, wherein the lipase is a thermostable lipase.
 13. The methodaccording to claim 11, wherein the lipase is derived from a strain ofHumicola species, Candida antarctica or Rhizomucor miehei.
 14. Themethod according to claim 11, wherein the ratio between the lipase andthe particulate silica carrier corresponds to an activity of at least100,000 LU/g of carrier (dry weight).
 15. The method according to claim10, wherein the silica has a purity of at least 50%.
 16. The methodaccording to claim 15, wherein the silica has a purity of at least 75%.17. The method according to claim 10, wherein the granulator is a highspeed mixer or a mixer granulator.
 18. The method of claim 10, whereinthe binder is gelatin or polyvinyl pyrrolidone.
 19. A method forinteresterification of fats, comprising contacting a fatty mixturecontaining a free fatty acid or a fatty acid ester with an immobilizedlipase preparation which is produced by:introducing a lipase and abinder in liquid form by atomization onto a particulate silica carrierhaving a particle size below 100 μm in a granulator, and simultaneouslycarrying out a granulation to form the immobilized lipase preparation.20. A method for interesterification of fats, comprising contacting afatty mixture containing a free fatty acid or a fatty acid ester with animmobilized lipase preparation which is produced by:contacting a liquidenzyme composition with a particulate silica carrier having a particlesize below 100 μm to obtain a particulate immobilized lipase preparationhaving a particle size below 100 μm, and introducing the particulateimmobilized enzyme preparation into a granulator, introducing a liquidcomprising a binder by atomization into the granulator, and carrying outa granulation.
 21. A method for the synthesis of a fatty acid estercomprising contacting an alcohol and a free fatty acid with animmobilized lipase preparation which is produced by:introducing a lipaseand a binder in liquid form by atomization onto a particulate silicacarrier having a particle size below 100 μm in a granulator, andsimultaneously carrying out a granulation to form the immobilized lipasepreparation.
 22. The method of claim 21, wherein the fatty acid ester isa glyceride and the alcohol is glycerol or a substituted glycerol.
 23. Amethod for the synthesis of a fatty acid ester comprising contacting analcohol and a free fatty acid with an immobilized lipase preparationwhich is produced by:contacting a liquid enzyme composition with aparticulate silica carrier having a particle size below 100 μm to obtaina particulate immobilized lipase preparation having a particle sizebelow 100 μm, and introducing the particulate immobilized enzymepreparation into a granulator, introducing a liquid comprising a binderby atomization into the granulator, and carrying out a granulation. 24.The method of claim 23, wherein the fatty acid ester is a glyceride andthe alcohol is glycerol or a substituted glycerol.